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ROCK activity regulates functional tight junction assembly during blastocyst formation in porcine parthenogenetic embryos.

ABSTRACT: The Rho-associated coiled-coil-containing protein serine/threonine kinases 1 and 2 (ROCK1 and ROCK2) are Rho subfamily GTPase downstream effectors that regulate cell migration, intercellular adhesion, cell polarity, and cell proliferation by stimulating actin cytoskeleton reorganization. Inhibition of ROCK proteins affects specification of the trophectoderm (TE) and inner cell mass (ICM) lineages, compaction, and blastocyst cavitation. However, the molecules involved in blastocyst formation are not known. Here, we examined developmental competence and levels of adherens/tight junction (AJ/TJ) constituent proteins, such as CXADR, OCLN, TJP1, and CDH1, as well as expression of their respective mRNAs, after treating porcine parthenogenetic four-cell embryos with Y-27632, a specific inhibitor of ROCK, at concentrations of 0, 10, 20, 100 µM for 24 h. Following this treatment, the blastocyst development rates were 39.1, 20.7, 10.0, and 0% respectively. In embryos treated with 20 µM treatment, expression levels of CXADR, OCLN, TJP1, and CDH1 mRNA and protein molecules were significantly reduced (P < 0.05). FITC-dextran uptake assay revealed that the treatment caused an increase in TE TJ permeability. Interestingly, the majority of the four-cell and morula embryos treated with 20 µM Y-27643 for 24 h showed defective compaction and cavitation. Taken together, our results indicate that ROCK activity may differentially affect assembly of AJ/TJs as well as regulate expression of genes encoding junctional proteins.

SUBMITTER: Kwon J

PROVIDER: S-EPMC4830244 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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ROCK activity regulates functional tight junction assembly during blastocyst formation in porcine parthenogenetic embryos.

Kwon Jeongwoo J Kim Nam-Hyung NH Choi Inchul I

PeerJ 20160411

The Rho-associated coiled-coil-containing protein serine/threonine kinases 1 and 2 (ROCK1 and ROCK2) are Rho subfamily GTPase downstream effectors that regulate cell migration, intercellular adhesion, cell polarity, and cell proliferation by stimulating actin cytoskeleton reorganization. Inhibition of ROCK proteins affects specification of the trophectoderm (TE) and inner cell mass (ICM) lineages, compaction, and blastocyst cavitation. However, the molecules involved in blastocyst formation are ...[more]

PMID: 27077008

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Project description:STUDY QUESTION:What is the impact of adenosine monophosphate-activated protein kinase (AMPK) activation on blastocyst formation, gene expression, and tight junction formation and function? SUMMARY ANSWER:AMPK activity must be tightly controlled for normal preimplantation development and blastocyst formation to occur. WHAT IS KNOWN ALREADY:AMPK isoforms are detectable in oocytes, cumulus cells and preimplantation embryos. Cultured embryos are subject to many stresses that can activate AMPK. STUDY DESIGN, SIZE, DURATION:Two primary experiments were carried out to determine the effect of AICAR treatment on embryo development and maintenance of the blastocoel cavity. Embryos were recovered from superovulated mice. First, 2-cell embryos were treated with a concentration series (0-2000 ?M) of AICAR for 48 h until blastocyst formation would normally occur. In the second experiment, expanded mouse blastocysts were treated for 9 h with 1000 ?M AICAR. PARTICIPANTS/MATERIALS, SETTING, METHODS:Outcomes measured included development to the blastocyst stage, cell number, blastocyst volume, AMPK phosphorylation, Cdx2 and blastocyst formation gene family expression (mRNAs and protein measured using quantitative RT-PCR, immunoblotting, immunofluorescence), tight junction function (FITC dextran dye uptake assay), and blastocyst ATP levels. The reversibility of AICAR treatment was assessed using Compound C (CC), a well-known inhibitor of AMPK, alone or in combination with AICAR. MAIN RESULTS AND THE ROLE OF CHANCE:Prolonged treatment with AICAR from the 2-cell stage onward decreases blastocyst formation, reduces total cell number, embryo diameter, leads to loss of trophectoderm cell contacts and membrane zona occludens-1 staining, and increased nuclear condensation. Treatment with CC alone inhibited blastocyst development only at concentrations that are higher than normally used. AICAR treated embryos displayed altered mRNA and protein levels of blastocyst formation genes. Treatment of blastocysts with AICAR for 9 h induced blastocyst collapse, altered blastocyst formation gene expression, increased tight junction permeability and decreased CDX2. Treated blastocysts displayed three phenotypes: those that were unaffected by treatment, those in which treatment was reversible, and those in which effects were irreversible. LARGE SCALE DATA:Not applicable. LIMITATIONS, REASONS FOR CAUTION:Our study investigates the effects of AICAR treatment on early development. While AICAR does increase AMPK activity and this is demonstrated in our study, AICAR is not a natural regulator of AMPK activity and some outcomes may result from off target non-AMPK AICAR regulated events. To support our results, blastocyst developmental outcomes were confirmed with two other well-known small molecule activators of AMPK, metformin and phenformin. WIDER IMPLICATIONS OF THE FINDINGS:Metformin, an AMPK activator, is widely used to treat type II diabetes and polycystic ovarian disorder (PCOS). Our results indicate that early embryonic AMPK levels must be tightly regulated to ensure normal preimplantation development. Thus, use of metformin should be carefully considered during preimplantation and early post-embryo transfer phases of fertility treatment cycles. STUDY FUNDING AND COMPETING INTEREST(S):Canadian Institutes of Health Research (CIHR) operating funds. There are no competing interests.

Dataset Information

ROCK activity regulates functional tight junction assembly during blastocyst formation in porcine parthenogenetic embryos.

Publications

ROCK activity regulates functional tight junction assembly during blastocyst formation in porcine parthenogenetic embryos.

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